Quinazoline based EGFR inhibitors

ABSTRACT

The present invention relates to quinazoline containing zinc-binding moiety based derivatives of formula I that have enhanced and unexpected properties as inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TK) and their use in the treatment of EGFR-TK related diseases and disorders such as cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/155,788 filed on Jun. 8, 2011, now U.S. Pat. No. 8,349,856, which isa continuation of U.S. application Ser. No. 11/852,474, now U.S. Pat.No. 7,977,347, filed on Sep. 10, 2007, which claims the benefit of U.S.application Ser. No. 60/843,644 filed on Sep. 11, 2006 and U.S.application Ser. No. 60/895,873 filed on Mar. 20, 2007. The entireteachings of the above applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor (EGFR, Erb-B1) belongs to a familyof proteins, involved in the proliferation of normal and malignant cells(Artega, C. L., J. Clin Oncol 19, 2001, 32-40). Overexpression ofEpidermal Growth Factor Receptor (EGFR) is present in at least 70% ofhuman cancers (Seymour, L. K., Curr Drug Targets 2, 2001, 117-133) suchas, non-small cell lung carcinomas (NSCLC), breast cancers, gliomas,squamous cell carcinoma of the head and neck, and prostate cancer(Raymond et al., Drugs 60 Suppl 1, 2000, discussion 41-2; Salomon etal., Crit. Rev Oncol Hematol 19, 1995, 183-232; Voldborg et al., AnnOncol 8, 1997, 1197-1206). The EGFR-TK is therefore widely recognized asan attractive target for the design and development of compounds thatcan specifically bind and inhibit the tyrosine kinase activity and itssignal transduction pathway in cancer cells, and thus can serve aseither diagnostic or therapeutic agents. For example, the EGFR tyrosinekinase (EGFR-TK) reversible inhibitor, TARCEVA®, is recently approved bythe FDA for treatment of NSCLC and advanced pancreatic cancer. Otheranti-EGFR targeted molecules have also been approved such as IRESSA®.

Despite the early success of Tarceva, it has become clear thatselectively targeting individual kinases can lead to the development ofdrug resistant tumors. Cells that have developed mutations within thedrug/kinase binding pocket display a growth advantage in the presence ofdrug eventually leading to disease progression. Accordingly, thediscovery and development of new molecules to target such kinases may beimportant to treat patients that have already developed resistance tocurrent therapeutic molecules.

SUMMARY OF THE INVENTION

The present invention relates to certain quinazolines that inhibit oneor more epidermal growth factor receptor tyrosine kinase (EGFR-TK), HDACor matrix metalloproteinase (MMP) and/or HER2 and are effective fortreating diseases related to EGFR-TK activity, HDAC activity and/or HER2activity, such as cancer and proliferative diseases.

Accordingly, the present invention provides a compound having thegeneral Formula I:

or its geometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs (e.g., esters) and solvatesthereof, wherein

-   -   X is O, S, CH₂, or —CONH—, preferably O;    -   B is an unsubstituted or hydroxy substituted C₃ to C₉ alkylene,        preferably an unsubstituted or hydroxy substituted straight        chain C₅ to C₇ alkylene, an unsubstituted or hydroxy substituted        most preferably a straight chain C₄ or C₆ alkylene, wherein B        can be a β-hydroxy alkylene;    -   R₁ is independently selected from hydrogen; hydroxy, C₁ to C₄        alkoxy, preferably methoxy; or substituted C₁ to C₄ alkoxy,        preferably C₁ to C₄ alkoxy substituted C₁ to C₄ alkoxy, such as        most preferably methoxyethoxy; and    -   R₂ is each independently selected from halogen (preferably Br,        Cl and F), hydroxy, C₁ to C₄ alkyl, C₂ to C₄ alkenyl, and C₂ to        C₄ alkynyl (preferably ethynyl);    -   n is 1, 2 or 3, preferably 1 or 2.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment of the compounds of the present invention arecompounds represented by formula (I) as illustrated above, or itsgeometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs and solvates thereof.

In a second embodiment of the compounds of the present invention arecompounds represented by formula (II) as illustrated below, or itsgeometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs and solvates thereof:

wherein B, R₁, R₂, and n are as previously defined.

In a third embodiment of the compounds of the present invention arecompounds represented by formula (III) as illustrated below, or itsgeometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs and solvates thereof:

wherein B and R₁ are as previously defined.

In a particularly preferred embodiment, the compound has the Formula IIIwherein R₁ is a methoxy and B is an unsubstituted or hydroxy substitutedstraight chain C₆ alkylene and pharmaceutically acceptable salts andprodrugs (e.g. esters) thereof.

In a fourth embodiment of the compounds of the present invention arecompounds represented by formula (IV) as illustrated below, or itsgeometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs and solvates thereof:

wherein B and R₁ are as previously defined.

In a fifth embodiment of the compounds of the present invention arecompounds represented by formula (V) as illustrated below, or itsgeometric isomers, enantiomers, diastereomers, racemates,pharmaceutically acceptable salts, prodrugs and solvates thereof:

In each of the above embodiments, R₁ is preferably hydrogen, hydroxy ormethoxy and, independently or collectively, B is preferably anunsubstituted or hydroxy substituted straight chain C₅ to C₇ alkylene,most preferably a straight chain C₆ alkylene.

In a particularly preferred embodiment, the compound has the Formula IVwherein R₁ is a methoxy and B is an unsubstituted or hydroxy substitutedstraight chain C₆ alkylene and pharmaceutically acceptable salts andprodrugs (e.g. esters) thereof.

The compounds according to the invention are metabolites of compoundsdescribed and claimed in the priority applications of this applicationand in related application U.S. application Ser. No. 11/852,604, thecontents of which are incorporated herein by reference.

The invention further provides methods for the prevention or treatmentof diseases or conditions involving aberrant proliferation,differentiation or survival of cells. In one embodiment, the inventionfurther provides for the use of one or more compounds of the inventionin the manufacture of a medicament for halting or decreasing diseasesinvolving aberrant proliferation, differentiation, or survival of cells.In preferred embodiments, the disease is cancer. In one embodiment, theinvention relates to a method of treating cancer in a subject in need oftreatment comprising administering to said subject a therapeuticallyeffective amount of a compound of the invention.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. For example, cancersinclude, but are not limited to, mesothelioma, leukemias and lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-celllymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, andmultiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma,adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronicmyeloid leukemia (CML), or hepatocellular carcinoma. Further examplesinclude myelodisplastic syndrome, childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular), lung cancer (e.g., small-cell and non small cell), breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, tumors related to Gorlin's syndrome (e.g.,medulloblastoma, meningioma, etc.), and liver cancer. Additionalexemplary forms of cancer which may be treated by the subject compoundsinclude, but are not limited to, cancer of skeletal or smooth muscle,stomach cancer, cancer of the small intestine, rectum carcinoma, cancerof the salivary gland, endometrial cancer, adrenal cancer, anal cancer,rectal cancer, parathyroid cancer, and pituitary cancer.

Additional cancers that the compounds described herein may be useful inpreventing, treating and studying are, for example, colon carcinoma,familiary adenomatous polyposis carcinoma and hereditary non-polyposiscolorectal cancer, or melanoma. Further, cancers include, but are notlimited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma,tongue carcinoma, salivary gland carcinoma, gastric carcinoma,adenocarcinoma, thyroid cancer (medullary and papillary thyroidcarcinoma, renal carcinoma, kidney parenchyma carcinoma, cervixcarcinoma, uterine corpus carcinoma, endometrium carcinoma, chorioncarcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumorssuch as glioblastoma, astrocytoma, meningioma, medulloblastoma andperipheral neuroectodermal tumors, gall bladder carcinoma, bronchialcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma, and plasmocytoma. In one aspect of the invention, thepresent invention provides for the use of one or more compounds of theinvention in the manufacture of a medicament for the treatment ofcancer.

In one embodiment, the present invention includes the use of one or morecompounds of the invention in the manufacture of a medicament thatprevents further aberrant proliferation, differentiation, or survival ofcells. For example, compounds of the invention may be useful inpreventing tumors from increasing in size or from reaching a metastaticstate. The subject compounds may be administered to halt the progressionor advancement of cancer or to induce tumor apoptosis or to inhibittumor angiogenesis. In addition, the instant invention includes use ofthe subject compounds to prevent a recurrence of cancer.

This invention further embraces the treatment or prevention of cellproliferative disorders such as hyperplasias, dysplasias andpre-cancerous lesions. Dysplasia is the earliest form of pre-cancerouslesion recognizable in a biopsy by a pathologist. The subject compoundsmay be administered for the purpose of preventing said hyperplasias,dysplasias or pre-cancerous lesions from continuing to expand or frombecoming cancerous. Examples of pre-cancerous lesions may occur in skin,esophageal tissue, breast and cervical intra-epithelial tissue.

“Combination therapy” includes the administration of the subjectcompounds in further combination with other biologically activeingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g. HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-1, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); Axl (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate non-kinase biological targets or processes. Such targetsinclude histone deacetylases (HDAC), DNA methyltransferase (DNMT), heatshock proteins (e.g. HSP90), and proteosomes.

In a preferred embodiment, subject compounds may be combined withantineoplastic agents (e.g. small molecules, monoclonal antibodies,antisense RNA, and fusion proteins) that inhibit one or more biologicaltargets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent,Sprycel, Nexavar, Sorafinib, CNF2024, RG108, BMS387032, Affinitak,Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD184322,Obatodax, ABT737 and AEE788. Such combinations may enhance therapeuticefficacy over efficacy achieved by any of the agents alone and mayprevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplastics such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA).

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude a 5HT₁ agonist, such as a triptan (e.g. sumatriptan ornaratriptan); an adenosine A1 agonist; an EP ligand; an NMDA modulator,such as a glycine antagonist; a sodium channel blocker (e.g.lamotrigine); a substance P antagonist (e.g. an NK₁ antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilising antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor .alpha.; an antibody therapy, such as a monoclonal antibodytherapy; an antiviral agent, such as a nucleoside inhibitor (e.g.lamivudine) or an immune system modulator (e.g. interferon); an opioidanalgesic; a local anaesthetic; a stimulant, including caffeine; anH₂-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g.omeprazole); an antacid (e.g. aluminum or magnesium hydroxide); anantiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine,phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine,naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine);an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, ordextramethorphan); a diuretic; or a sedating or non-sedatingantihistamine.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutralendopeptidases collectively capable of degrading essentially all matrixcomponents. Over 20 MMP modulating agents are in pharmaceutical develop,almost half of which are indicated for cancer. The University of Torontoresearchers have reported that HDACs regulate MMP expression andactivity in 3T3 cells. In particular, inhibition of HDAC by trichostatinA (TSA), which has been shown to prevent tumorigenesis and metastasis,decreases mRNA as well as zymographic activity of gelatinase A (MMP2;Type IV collagenase), a matrix metalloproteinase, which is itself,implicated in tumorigenesis and metastasis (Ailenberg M., Silverman M.,Biochem Biophys Res Commun. 2002, 298:110-115). Another recent articlethat discusses the relationship of HDAC and MMPs can be found in YoungD. A., et al., Arthritis Research & Therapy, 2005, 7: 503. Furthermore,the commonality between HDAC and MMPs inhibitors is their zinc-bindingfunctionality. Therefore, in one aspect of the invention, compounds ofthe invention can be used as MMP inhibitors and may be of use in thetreatment of disorders relating to or associated with dysregulation ofMMP. The overexpression and activation of MMPs are known to inducetissue destruction and are also associated with a number of specificdiseases including rheumatoid arthritis, periodontal disease, cancer andatherosclerosis.

The compounds may also be used in the treatment of a disorder involving,relating to or, associated with dysregulation of histone deacetylase(HDAC). There are a number of disorders that have been implicated by orknown to be mediated at least in part by HDAC activity, where HDACactivity is known to play a role in triggering disease onset, or whosesymptoms are known or have been shown to be alleviated by HDACinhibitors. Disorders of this type that would be expected to be amenableto treatment with the compounds of the invention include the followingbut not limited to: Anti-proliferative disorders (e.g. cancers);Neurodegenerative diseases including Huntington's Disease, Polyglutaminedisease, Parkinson's Disease, Alzheimer's Disease, Seizures,Striatonigral degeneration, Progressive supranuclear palsy, Torsiondystonia, Spasmodic torticollis and dyskinesis, Familial tremor, Gillesde la Tourette syndrome, Diffuse Lewy body disease, Progressivesupranuclear palsy, Pick's disease, intracerebral hemorrhage, Primarylateral sclerosis, Spinal muscular atrophy, Amyotrophic lateralsclerosis, Hypertrophic interstitial polyneuropathy, Retinitispigmentosa, Hereditary optic atrophy, Hereditary spastic paraplegia,Progressive ataxia and Shy-Drager syndrome; Metabolic diseases includingType 2 diabetes; Degenerative Diseases of the Eye including Glaucoma,Age-related macular degeneration, Rubeotic glaucoma; Inflammatorydiseases and/or Immune system disorders including Rheumatoid Arthritis(RA), Osteoarthritis, Juvenile chronic arthritis, Graft versus Hostdisease, Psoriasis, Asthma, Spondyloarthropathy, Crohn's Disease,inflammatory bowel disease Colitis Ulcerosa, Alcoholic hepatitis,Diabetes, Sjoegrens's syndrome, Multiple Sclerosis, Ankylosingspondylitis, Membranous glomerulopathy, Discogenic pain, Systemic LupusErythematosus; Disease involving angiogenesis including cancer,psoriasis, rheumatoid arthritis; Psychological disorders includingbipolar disease, schizophrenia, mania, depression and dementia;Cardiovascular Diseases including heart failure, restenosis andarteriosclerosis; Fibrotic diseases including liver fibrosis, cysticfibrosis and angiofibroma; Infectious diseases including Fungalinfections, such as Candida Albicans, Bacterial infections, Viralinfections, such as Herpes Simplex, Protozoal infections, such asMalaria, Leishmania infection, Trypanosoma brucei infection,Toxoplasmosis and coccidlosis and Haematopoietic disorders includingthalassemia, anemia and sickle cell anemia.

In one embodiment, compounds of the invention can be used to induce orinhibit apoptosis, a physiological cell death process critical fornormal development and homeostasis. Alterations of apoptotic pathwayscontribute to the pathogenesis of a variety of human diseases. Compoundsof the invention, as modulators of apoptosis, will be useful in thetreatment of a variety of human diseases with abberations in apoptosisincluding cancer (particularly, but not limited to, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis), viral infections (including, but notlimited to, herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus andadenovirus), autoimmune diseases (including, but not limited to,systemic lupus, erythematosus, immune mediated glomerulonephritis,rheumatoid arthritis, psoriasis, inflammatory bowel diseases, andautoimmune diabetes mellitus), neurodegenerative disorders (including,but not limited to, Alzheimer's disease, AIDS-related dementia,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration), AIDS,myelodysplastic syndromes, aplastic anemia, ischemic injury associatedmyocardial infarctions, stroke and reperfusion injury, arrhythmia,atherosclerosis, toxin-induced or alcohol induced liver diseases,hematological diseases (including, but not limited to, chronic anemiaand aplastic anemia), degenerative diseases of the musculoskeletalsystem (including, but not limited to, osteoporosis and arthritis),aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis,kidney diseases, and cancer pain.

In one aspect, the invention provides the use of compounds of theinvention for the treatment and/or prevention of immune response orimmune-mediated responses and diseases, such as the prevention ortreatment of rejection following transplantation of synthetic or organicgrafting materials, cells, organs or tissue to replace all or part ofthe function of tissues, such as heart, kidney, liver, bone marrow,skin, cornea, vessels, lung, pancreas, intestine, limb, muscle, nervetissue, duodenum, small-bowel, pancreatic-islet-cell, includingxeno-transplants, etc.; to treat or prevent graft-versus-host disease,autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis,myasthenia gravis, type I diabetes uveitis, juvenile-onset orrecent-onset diabetes mellitus, uveitis, Graves disease, psoriasis,atopic dermatitis, Crohn's disease, ulcerative colitis, vasculitis,auto-antibody mediated diseases, aplastic anemia, Evan's syndrome,autoimmune hemolytic anemia, and the like; and further to treatinfectious diseases causing aberrant immune response and/or activation,such as traumatic or pathogen induced immune disregulation, includingfor example, that which are caused by hepatitis B and C infections, HIV,staphylococcus aureus infection, viral encephalitis, sepsis, parasiticdiseases wherein damage is induced by an inflammatory response (e.g.,leprosy); and to prevent or treat circulatory diseases, such asarteriosclerosis, atherosclerosis, vasculitis, polyarteritis nodosa andmyocarditis. In addition, the present invention may be used toprevent/suppress an immune response associated with a gene therapytreatment, such as the introduction of foreign genes into autologouscells and expression of the encoded product. Thus in one embodiment, theinvention relates to a method of treating an immune response disease ordisorder or an immune-mediated response or disorder in a subject in needof treatment comprising administering to said subject a therapeuticallyeffective amount of a compound of the invention.

In one aspect, the invention provides the use of compounds of theinvention in the treatment of a variety of neurodegenerative diseases, anon-exhaustive list of which includes: I. Disorders characterized byprogressive dementia in the absence of other prominent neurologic signs,such as Alzheimer's disease; Senile dementia of the Alzheimer type; andPick's disease (lobar atrophy); II. Syndromes combining progressivedementia with other prominent neurologic abnormalities such as A)syndromes appearing mainly in adults (e.g., Huntington's disease,Multiple system atrophy combining dementia with ataxia and/ormanifestations of Parkinson's disease, Progressive supranuclear palsy(Steel-Richardson-Olszewski), diffuse Lewy body disease, andcorticodentatonigral degeneration); and B) syndromes appearing mainly inchildren or young adults (e.g., Hallervorden-Spatz disease andprogressive familial myoclonic epilepsy); III. Syndromes of graduallydeveloping abnormalities of posture and movement such as paralysisagitans (Parkinson's disease), striatonigral degeneration, progressivesupranuclear palsy, torsion dystonia (torsion spasm; dystonia musculorumdeformans), spasmodic torticollis and other dyskinesis, familial tremor,and Gilles de la Tourette syndrome; IV. Syndromes of progressive ataxiasuch as cerebellar degenerations (e.g., cerebellar cortical degenerationand olivopontocerebellar atrophy (OPCA)); and spinocerebellardegeneration (Friedreich's atazia and related disorders); V. Syndrome ofcentral autonomic nervous system failure (Shy-Drager syndrome); VI.Syndromes of muscular weakness and wasting without sensory changes(motorneuron disease such as amyotrophic lateral sclerosis, spinalmuscular atrophy (e.g., infantile spinal muscular atrophy(Werdnig-Hoffman), juvenile spinal muscular atrophy(Wohlfart-Kugelberg-Welander) and other forms of familial spinalmuscular atrophy), primary lateral sclerosis, and hereditary spasticparaplegia; VII. Syndromes combining muscular weakness and wasting withsensory changes (progressive neural muscular atrophy; chronic familialpolyneuropathies) such as peroneal muscular atrophy(Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy(Dejerine-Sottas), and miscellaneous forms of chronic progressiveneuropathy; VIII. Syndromes of progressive visual loss such aspigmentary degeneration of the retina (retinitis pigmentosa), andhereditary optic atrophy (Leber's disease). Furthermore, compounds ofthe invention can be implicated in chromatin remodeling.

The invention encompasses pharmaceutical compositions comprisingpharmaceutically acceptable salts of the compounds of the invention asdescribed above. Examples of suitable salts include but are not limitedto the hydrochloride, citrate or tartrate salt, preferably the tartratesalt. The invention also encompasses pharmaceutical compositionscomprising solvates or hydrates of the compounds of the invention. Theterm “hydrate” includes but is not limited to hemihydrate, monohydrate,dihydrate, trihydrate and the like.

The invention further encompasses pharmaceutical compositions comprisingany solid or liquid physical form of the compound of the invention. Forexample, the compounds can be in a crystalline form, in amorphous form,and have any particle size. The particles may be micronized, or may beagglomerated, particulate granules, powders, oils, oily suspensions orany other form of solid or liquid physical form.

The compounds of the invention, and derivatives, fragments, analogs,homologs, pharmaceutically acceptable salts or hydrate thereof can beincorporated into pharmaceutical compositions suitable foradministration, together with a pharmaceutically acceptable carrier orexcipient. Such compositions typically comprise a therapeuticallyeffective amount of any of the compounds above, and a pharmaceuticallyacceptable carrier. Preferably, the effective amount when treatingcancer is an amount effective to selectively induce terminaldifferentiation of suitable neoplastic cells and less than an amountwhich causes toxicity in a patient.

Compounds of the invention may be administered by any suitable means,including, without limitation, parenteral, intravenous, intramuscular,subcutaneous, implantation, oral, sublingual, buccal, nasal, pulmonary,transdermal, topical, vaginal, rectal, and transmucosal administrationsor the like. Topical administration can also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. Pharmaceutical preparations include a solid, semisolid orliquid preparation (tablet, pellet, troche, capsule, suppository, cream,ointment, aerosol, powder, liquid, emulsion, suspension, syrup,injection etc.) containing a compound of the invention as an activeingredient, which is suitable for selected mode of administration. Inone embodiment, the pharmaceutical compositions are administered orally,and are thus formulated in a form suitable for oral administration,i.e., as a solid or a liquid preparation. Suitable solid oralformulations include tablets, capsules, pills, granules, pellets,sachets and effervescent, powders, and the like. Suitable liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like. In one embodiment of the present invention, thecomposition is formulated in a capsule. In accordance with thisembodiment, the compositions of the present invention comprise inaddition to the active compound and the inert carrier or diluent, a hardgelatin capsule.

Any inert excipient that is commonly used as a carrier or diluent may beused in the formulations of the present invention, such as for example,a gum, a starch, a sugar, a cellulosic material, an acrylate, ormixtures thereof. A preferred diluent is microcrystalline cellulose. Thecompositions may further comprise a disintegrating agent (e.g.,croscarmellose sodium) and a lubricant (e.g., magnesium stearate), andmay additionally comprise one or more additives selected from a binder,a buffer, a protease inhibitor, a surfactant, a solubilizing agent, aplasticizer, an emulsifier, a stabilizing agent, a viscosity increasingagent, a sweetener, a film forming agent, or any combination thereof.Furthermore, the compositions of the present invention may be in theform of controlled release or immediate release formulations.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Solutions or suspensions can also include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

In addition, the compositions may further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), buffers (e.g., tris-HCl., acetate,phosphate) of various pH and ionic strength, additives such as albuminor gelatin to prevent absorption to surfaces, detergents (e.g., Tween20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol,cyclodextrins), a glidant (e.g., colloidal silicon dioxide),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylatedhydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose,hydroxypropylmethyl cellulose), viscosity increasing agents (e.g.,carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents(e.g., peppermint, methyl salicylate, or orange flavoring),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g., stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates)and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

In one preferred embodiment, the compound can be formulated in anaqueous solution for intravenous injection. In one embodiment,solubilizing agents can be suitably employed. A particularly preferredsolubilizing agent includes cyclodextrins and modified cyclodextrins,such as sulfonic acid substituted β-cyclodextrin derivative or saltthereof. An example of such a solubilizing agent is sold under thetrademark CAPTISOL® by CyDex, Inc. CAPTISOL® is a polyanionicβ-cyclodextrin derivative with a sodium sulfonate salt separated fromthe lipophilic cavity by a butyl ether spacer group, or sulfobutylether(SBE). The selection of the SBE7-β-CD as the cyclodextrin with the mostdesirable safety profile and drug carrier properties is based uponevaluations of the mono, tetra and hepta-substituted preparations (SBE1,SBE4, and SBE7). CAPTISOL® is the trade name for CyDex's SBE7-β-CDPRODUCT.

Relative to β-cyclodextrin, the preferred solubilizing agents, such asCAPTISOL®, provide superior water solubility in excess of 70, preferably90 grams/100 ml.

In one embodiment, the solubilizing agent is added to the aqueoussolution in an amount of at least 15% weight/volume, preferably about30% weight/volume. Additional optional excipients can include dextran inan amount of at least about 1% weight/volume, preferably about 5%weight/volume.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Daily administration may be repeated continuously for a period ofseveral days to several years. Oral treatment may continue for betweenone week and the life of the patient. Preferably the administration maytake place for five consecutive days after which time the patient can beevaluated to determine if further administration is required. Theadministration can be continuous or intermittent, e.g., treatment for anumber of consecutive days followed by a rest period. The compounds ofthe present invention may be administered intravenously on the first dayof treatment, with oral administration on the second day and allconsecutive days thereafter.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than anamount that would cause toxicity in the patient. In certain embodiments,the amount of the compound that is administered to the patient is lessthan the amount that causes a concentration of the compound in thepatient's plasma to equal or exceed the toxic level of the compound.Preferably, the concentration of the compound in the patient's plasma ismaintained at about 10 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 25 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 50 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 100 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 1000 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 2500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 5000 nM. Theoptimal amount of the compound that should be administered to thepatient in the practice of the present invention will depend on theparticular compound used and the type of cancer being treated.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl” embraces linear or branched radicals having one toabout twenty carbon atoms or, preferably, one to about twelve carbonatoms. More preferred alkyl radicals are “lower alkyl” radicals havingone to about ten carbon atoms. Most preferred are lower alkyl radicalshaving one to about eight carbon atoms. Examples of such radicalsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twenty carbon atoms or,preferably, two to about twelve carbon atoms. More preferred alkenylradicals are “lower alkenyl” radicals having two to about ten and morepreferably about two to about eight carbon atoms. Examples of alkenylradicals include ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl.The terms “alkenyl”, and “lower alkenyl”, embrace radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.

The term “alkynyl” embraces linear or branched radicals having at leastone carbon-carbon triple bond of two to about twenty carbon atoms or,preferably, two to about twelve carbon atoms. More preferred alkynylradicals are “lower alkynyl” radicals having two to about ten and morepreferably two to about eight carbon atoms. Examples of alkynyl radicalsinclude propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl and1-pentynyl.

The term “alkoxy” embraces linear or branched oxy-containing radicalseach having alkyl portions of one to about twenty carbon atoms or,preferably, one to about twelve carbon atoms. More preferred alkoxyradicals are “lower alkoxy” radicals having one to about ten and morepreferably having one to about eight carbon atoms. Examples of suchradicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.

The term “alkoxyalkoxy” embraces alkoxy radicals having one or morealkoxy radicals attached to the alkyl radical, that is, to formmonoalkoxyalkyl and dialkoxyalkyl radicals.

The term “compound” is defined herein to include pharmaceuticallyacceptable salts, solvates, hydrates, polymorphs, enantiomers,diastereoisomers, racemates and the like of the compounds having aformula as set forth herein.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent can be further substituted.

The terms “halogen” or “halo” as used herein, refers to an atom selectedfrom fluorine, chlorine, bromine and iodine.

As used herein, the term “aberrant proliferation” refers to abnormalcell growth.

The phrase “adjunctive therapy” encompasses treatment of a subject withagents that reduce or avoid side effects associated with the combinationtherapy of the present invention, including, but not limited to, thoseagents, for example, that reduce the toxic effect of anticancer drugs,e.g., bone resorption inhibitors, cardioprotective agents; prevent orreduce the incidence of nausea and vomiting associated withchemotherapy, radiotherapy or operation; or reduce the incidence ofinfection associated with the administration of myelosuppressiveanticancer drugs.

The term “angiogenesis,” as used herein, refers to the formation ofblood vessels. Specifically, angiogenesis is a multi-step process inwhich endothelial cells focally degrade and invade through their ownbasement membrane, migrate through interstitial stroma toward anangiogenic stimulus, proliferate proximal to the migrating tip, organizeinto blood vessels, and reattach to newly synthesized basement membrane(see Folkman et al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).Anti-angiogenic agents interfere with this process. Examples of agentsthat interfere with several of these steps include thrombospondin-1,angiostatin, endostatin, interferon alpha and compounds such as matrixmetalloproteinase (MMP) inhibitors that block the actions of enzymesthat clear and create paths for newly forming blood vessels to follow;compounds, such as .alpha.v.beta 3 inhibitors, that interfere withmolecules that blood vessel cells use to bridge between a parent bloodvessel and a tumor; agents, such as specific COX-2 inhibitors, thatprevent the growth of cells that form new blood vessels; andprotein-based compounds that simultaneously interfere with several ofthese targets.

The term “apoptosis” as used herein refers to programmed cell death assignaled by the nuclei in normally functioning human and animal cellswhen age or state of cell health and condition dictates. An “apoptosisinducing agent” triggers the process of programmed cell death.

The term “cancer” as used herein denotes a class of diseases ordisorders characterized by uncontrolled division of cells and theability of these cells to invade other tissues, either by direct growthinto adjacent tissue through invasion or by implantation into distantsites by metastasis.

The term “devices” refers to any appliance, usually mechanical orelectrical, designed to perform a particular function.

As used herein, the term “dysplasia” refers to abnormal cell growth andtypically refers to the earliest form of pre-cancerous lesionrecognizable in a biopsy by a pathologist.

As used herein, the term “effective amount of the subject compounds,”with respect to the subject method of treatment, refers to an amount ofthe subject compound which, when delivered as part of desired doseregimen, brings about, e.g. a change in the rate of cell proliferationand/or state of differentiation and/or rate of survival of a cell toclinically acceptable standards. This amount may further relieve to someextent one or more of the symptoms of a neoplasia disorder, including,but is not limited to: 1) reduction in the number of cancer cells; 2)reduction in tumor size; 3) inhibition (i.e., slowing to some extent,preferably stopping) of cancer cell infiltration into peripheral organs;3) inhibition (i.e., slowing to some extent, preferably stopping) oftumor metastasis; 4) inhibition, to some extent, of tumor growth; 5)relieving or reducing to some extent one or more of the symptomsassociated with the disorder; and/or 6) relieving or reducing the sideeffects associated with the administration of anticancer agents.

The term “hyperplasia,” as used herein, refers to excessive celldivision or growth.

The phrase an “immunotherapeutic agent” refers to agents used totransfer the immunity of an immune donor, e.g., another person or ananimal, to a host by inoculation. The term embraces the use of serum orgamma globulin containing performed antibodies produced by anotherindividual or an animal; nonspecific systemic stimulation; adjuvants;active specific immunotherapy; and adoptive immunotherapy. Adoptiveimmunotherapy refers to the treatment of a disease by therapy or agentsthat include host inoculation of sensitized lymphocytes, transferfactor, immune RNA, or antibodies in serum or gamma globulin.

The term “inhibition,” in the context of neoplasia, tumor growth ortumor cell growth, may be assessed by delayed appearance of primary orsecondary tumors, slowed development of primary or secondary tumors,decreased occurrence of primary or secondary tumors, slowed or decreasedseverity of secondary effects of disease, arrested tumor growth andregression of tumors, among others. In the extreme, complete inhibition,is referred to herein as prevention or chemoprevention.

The term “metastasis,” as used herein, refers to the migration of cancercells from the original tumor site through the blood and lymph vesselsto produce cancers in other tissues. Metastasis also is the term usedfor a secondary cancer growing at a distant site.

The term “neoplasm,” as used herein, refers to an abnormal mass oftissue that results from excessive cell division. Neoplasms may bebenign (not cancerous), or malignant (cancerous) and may also be calleda tumor. The term “neoplasia” is the pathological process that resultsin tumor formation.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “proliferation” refers to cells undergoing mitosis.

The phrase “EGFR-TK related disease or disorder” refers to a disease ordisorder characterized by inappropriate EGFR-TK activity orover-activity of the EGFR-TK. Inappropriate activity refers to either:(i) EGFR-TK expression in cells which normally do not express EGFR-TKs;(ii) increased EGFR-TK expression leading to unwanted cellproliferation, differentiation and/or growth; or, (iii) decreasedEGFR-TK expression leading to unwanted reductions in cell proliferation,differentiation and/or growth. Over-activity of EGFR-TKs refers toeither amplification of the gene encoding a particular EGFR-TK orproduction of a level of EGFR-TK activity which can correlate with acell proliferation, differentiation and/or growth disorder (that is, asthe level of the EGFR-TK increases, the severity of one or more of thesymptoms of the cellular disorder increases). Over activity can also bethe result of ligand independent or constitutive activation as a resultof mutations such as deletions of a fragment of an EGFR-TK responsiblefor ligand binding.

The phrase a “radio therapeutic agent” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.

The term “recurrence” as used herein refers to the return of cancerafter a period of remission. This may be due to incomplete removal ofcells from the initial cancer and may occur locally (the same site ofinitial cancer), regionally (in vicinity of initial cancer, possibly inthe lymph nodes or tissue), and/or distally as a result of metastasis.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein a mammal, including a human being, issubject to medical aid with the object of improving the mammal'scondition, directly or indirectly.

The term “vaccine” includes agents that induce the patient's immunesystem to mount an immune response against the tumor by attacking cellsthat express tumor associated antigens (Teas).

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid or inorganic acid. Examples of pharmaceuticallyacceptable nontoxic acid addition salts include, but are not limited to,salts of an amino group formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloricacid or with organic acids such as acetic acid, maleic acid, tartaricacid, citric acid, succinic acid lactobionic acid or malonic acid or byusing other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include, but are not limited to,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters which hydrolyze in vivo and include those that break down readilyin the human body to leave the parent compound or a salt thereof.Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of thepresent invention. “Prodrug”, as used herein means a compound which isconvertible in vivo by metabolic means (e.g. by hydrolysis) to acompound of the invention. Various forms of prodrugs are known in theart, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs,Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4,Academic Press (1985); Krogsgaard-Larsen, et al. (ed.) “Design andApplication of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug DeliverReviews, 8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975); and Bernard Testa &Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002). Examplesof particularly preferred prodrugs include esters of the carboxylicacids of the invention. Preferred esters include aliphatic esters (e.g.,alkyl, such as lower alkyl esters) and aromatic esters (such as phenylesters). Other prodrugs include derivatives of the acid group that canbe hydrolyzed in vivo.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, such as sterilepyrogen-free water. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “subject” as used herein refers to an animal. Preferably theanimal is a mammal. More preferably the mammal is a human. A subjectalso refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, fish, birds and the like.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and may include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds described herein may contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions(John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers and/or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers or excipients.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;cyclodextrins such as alpha-(α), beta-(B) and gamma-(γ) cyclodextrins;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as solubilizingagents, coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid; b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate; h) absorbents such as kaolinand bentonite clay; and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

For pulmonary delivery, a therapeutic composition of the invention isformulated and administered to the patient in solid or liquidparticulate form by direct administration e.g., inhalation into therespiratory system. Solid or liquid particulate forms of the activecompound prepared for practicing the present invention include particlesof respirable size: that is, particles of a size sufficiently small topass through the mouth and larynx upon inhalation and into the bronchiand alveoli of the lungs. Delivery of aerosolized therapeutics,particularly aerosolized antibiotics, is known in the art (see, forexample U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No.5,508,269 to Smith et al., and WO 98/43650 by Montgomery, all of whichare incorporated herein by reference). A discussion of pulmonarydelivery of antibiotics is also found in U.S. Pat. No. 6,014,969,incorporated herein by reference.

By a “therapeutically effective amount” of a compound of the inventionis meant an amount of the compound which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). An effective amount of the compounddescribed above may range from about 0.1 mg/Kg to about 500 mg/Kg,preferably from about 1 to about 50 mg/Kg. Effective doses will alsovary depending on route of administration, as well as the possibility ofco-usage with other agents. It will be understood, however, that thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or contemporaneously with thespecific compound employed; and like factors well known in the medicalarts.

The total daily dose of the compounds of this invention administered toa human or other animal in single or in divided doses can be in amounts,for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1to 25 mg/kg body weight. Single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. In general,treatment regimens according to the present invention compriseadministration to a patient in need of such treatment from about 10 mgto about 1000 mg of the compound(s) of this invention per day in singleor multiple doses.

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.1 toabout 500 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with pharmaceutically excipients or carriers toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Alternatively,such preparations may contain from about 20% to about 80% activecompound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

Synthetic Methods

A quinazoline derivative of the formula I, or apharmaceutically-acceptable salt thereof, may be prepared by any processknown to be applicable to the preparation of chemically-relatedcompounds. Suitable processes for making certain intermediates include,for example, those illustrated in European Patent Applications Nos.0520722, 0566226, 0602851, 0635498, 0635507, U.S. Pat. Nos. 5,457,105,5,770,599, US Publication No. 2003/0158408 and reference such as, J.Med. Chem. 2004, 47, 871-887. Necessary starting materials may beobtained by standard procedures of organic chemistry. The preparation ofsuch starting materials is described within the accompanyingnon-limiting Examples. Alternatively necessary starting materials areobtainable by analogous procedures to those illustrated which are withinthe ordinary skill of a chemist.

The compounds and processes of the present invention will be betterunderstood in connection with the following representative syntheticschemes that illustrate the methods by which the compounds of theinvention may be prepared, which are intended as an illustration onlyand not limiting of the scope of the invention. For simplicity, thescheme numbering preserves the numbering used in the priorityapplications.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1 Preparation of4-(3-Chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ol (Compound0109) Step 1a. 6,7-Dimethoxyquinazolin-4(3H)-one (Compound 0102)

A mixture of methyl 2-amino-4,5-dimethoxybenzoic acid 0101 (2.1 g, 10mmol), ammonium formate (0.63 g, 10 mmol) and formamide (7 ml) wasstirred and heated to 190˜200° C. for 2 hours. Then the mixture wascooled to room temperature. The precipitate was isolated, washed withwater and dried to provide the title compound 0102 as a brown solid (1.8g, 84.7%): LCMS: m/z 207[M+1]⁺; ¹H NMR (DMSO) δ 3.87 (s, 3H), 3.89 (s,3H), 7.12 (s, 1H), 7.43 (s, 1H), 7.97 (s, 1H), 12.08 (bs, 1H).

Step 1b. 6-Hydroxy-7-methoxyquinazolin-4(3H)-one (Compound 0103)

6,7-Dimethoxyquinazolin-4(3H)-one (0102) (10.3 g, 50 mmol) was addedportionwise to stirred methanesulphonic acid (68 ml). L-Methionone (8.6g, 57.5 mmol) was then added and resultant mixture was heated to150˜160° C. for 5 hours. The mixture was cooled to room temperature andpoured onto a mixture (250 ml) of ice and water. The mixture wasneutralized by the addition of aqueous sodium hydroxide solution (40%).The precipitate was isolated, washed with water and dried to yield titlecompound 0103 as a grey solid (10 g, crude): LCMS: m/z 193[M+1]⁺.

Step 1c. 3,4-Dihydro-7-methoxy-4-oxoquinazolin-6-yl acetate (Compound0104)

A mixture of 6-hydroxy-7-methoxyquinazolin-4(3H)-one (0103) (10 gcrude), acetic anhydride (100 ml) and pyridine (8 ml) was stirred andheated to reflux for 3 hours. The mixture was cooled to room temperatureand poured into a mixture (250 ml) of ice and water. The precipitate wasisolated and dried to yield the title product 0104 as a grey solid (5.8g, 50% two step overall yield): LCMS: m/z 235[M+1]⁺; ¹H NMR (CDCl₃) δ2.27 (s, 3H), 3.89 (s, 3H), 7.28 (s, 1H), 7.72 (s, 1H), 8.08 (d, 1H),12.20 (bs, 1H).

Step 1d. 4-Chloro-7-methoxyquinazolin-6-yl acetate (Compound 0105)

A mixture of 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-yl acetate (0104)(2.0 g, 8.5 mmol) and phosphoryl trichloride (20 ml) was stirred andheated to reflux for 3 hours. When a clear solution was obtained, theexcessive phosphoryl trichloride was removed under reduced pressure. Theresidue was dissolved in dichloromethane (50 ml) and the organic layerwas washed with aqueous NaHCO₃ solution (20 ml×2) and brine (20 ml×1)and dried over MgSO₄, filtered and evaporated to give the title product0105 as a yellow solid (1.4 g, 65%): LCMS: m/z 249[M+1]⁺; ¹H NMR (CDCl₃)δ 2.40 (s, 3H), 4.03 (s, 3H), 7.44 (s, 1H), 7.90 (s, 1H), 8.95 (bs, 1H).

Step 1e. 4-(3-Chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ylacetate hydrochloride (Compound 0108)

A mixture of 4-chloro-7-methoxyquinazolin-6-yl acetate (0105) (1.3 g,5.1 mmol) and 3-chloro-4-fluorobenzenamine 0106 (1.5 g, 10.2 mmol) inisopropanol (45 ml) was stirred and heated to reflux for 3 hours. Themixture was cooled to room temperature and resulting precipitate wasisolated. The solid was then dried to give the title compound 0108 as alight yellow solid (1.6 g, 79%): LCMS: m/z 362[M+1]⁺; ¹H NMR (DMSO) δ2.36 (s, 3H), 3.98 (s, 3H), 7.49 (s, 1H), 7.52 (d, 1H), 7.72 (m, 1H),8.02 (dd, 1H), 8.71 (s, 1H), 8.91 (s, 1H), 11.4 (bs, 1H).

Step 1f. 4-(3-Chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ol(Compound 0109)

A mixture of compound (0107) (1.41 g, 3.5 mmol), LiOH H₂O (0.5 g, 11.7mmol) in methanol (100 ml) and H₂O (100 ml) was stirred at roomtemperature for 0.5 hour. The mixture was neutralized by addition ofdilution acetic acid. The precipitate was isolated and dried to give thetitle compound 0109 as a grey solid (1.06 g, 94%): LCMS: m/z 320[M+1]⁺;¹H NMR (DMSO) δ 3.99 (s, 3H), 7.20 (s, 1H), 7.38 (t, 1H), 7.75 (s, 1H),7.81 (m, 1H), 8.20 (m, 1H), 8.46 (s, 1H), 9.46 (s, 1H), 9.68 (s, 1H).

Step 1g. Ethyl2-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)acetate(Compound 0110-1)

A mixture of compound 0109 (300 mg, 0.94 mmol) and Ethyl 2-bromoacetate(163 mg, 0.98 mmol) and potassium carbonate (323 mg, 2.35 mmol) inN,N-dimethylformamide (6 ml) was stirred and heated to 40° for 30minutes. The reaction process was monitored by TLC. The mixture wasfiltrated. The filtration was concentrated under reduce pressure. Theresidues was wash with diethyl ether and dried to give the titlecompound 0110-1 as a yellow solid (280 mg, 74%): LCMS: m/z 406[M+1]⁺; ¹HNMR (DMSO) δ 1.23 (t, 3H), 3.96 (s, 3H), 4.20 (q, 2H), 4.95 (s, 2H),7.24 (s, 1H), 7.44 (t, 1H), 7.75 (m, 1H), 7.82 (s, 1H), 8.10 (dd, 1H),8.51 (s, 1H), 9.54 (s, 1H).

Example 2 Preparation of Ethyl4-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate(Compound 0110-3) Step 2a. Ethyl4-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate(Compound 0110-3)

The title compound 0110-3 was prepared as a yellow solid (220 mg, 80.5%)from compound 0109 from step 1f (200 mg, 0.63 mmol) and ethyl4-bromobutyrate (135 mg, 0.69 mmol) using a procedure similar to thatdescribed for compound 0110-1 (example 1): LCMS: m/z 434[M+1]⁺; ¹H NMR(CDCl₃) δ 1.36 (t, 3H), 2.23 (m, 2H), 2.57 (t, 2H), 4.03 (s, 3H), 4.32(m, 4H), 7.15 (t, 1H), 7.25 (m, 1H), 7.87 (s, 1H), 8.00 (m, 2H), 8.15(bs, 1H), 8.57 (s, 1H).

Example 3 Preparation of Ethyl6-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate(Compound 0110-5) Step 3a. Ethyl6-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate(Compound 0110-5)

The title compound 0110-5 was prepared as a yellow solid (510 mg, 68%)from compound 0109 from step 1f (510 mg, 1.6 mmol) and ethyl6-bromohexanoate (430 mg, 1.9 mmol) using a procedure similar to thatdescribed for compound 0110-1 (Example 1): LCMS: m/z 462[M+1]⁺; ¹H NMR(CDCl₃): δ 1.24 (t, 3H), 1.55 (m, 2H), 1.74 (m, 2H), 1.91 (m, 2H), 2.38(m, 2H), 3.97 (s, 3H), 4.13 (m, 4H), 7.15 (t, 1H), 7.25 (m, 2H), 7.60(m, 1H), 7.86 (m, 1H), 7.91 (dd, 1H), 8.61 (s, 1H).

Example 4 Preparation of Ethyl7-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate(compound 0110-6) Step 4a. Ethyl7-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate(compound 0110-6)

The title compound 0110-6 was prepared as a yellow solid (390 mg, 53%)from compound 0109 from step 1f (512 mg, 1.6 mmol) and ethyl7-bromoheptanoate (438 mg, 1.8 mmol) using a procedure similar to thatdescribed for compound 0110-1 (Example 1): LCMS: m/z 476[M+1]⁺; ¹H NMR(CDCl₃) δ 1.24 (t, 3H), 1.43 (m, 4H), 1.66 (m, 2H), 1.88 (m, 2H), 2.32(t, 2H), 3.97 (s, 3H), 4.07 (t, 2H), 4.12 (q, 2H), 7.15 (t, 1H), 7.23(t, 2H), 7.66 (m, 1H), 7.75 (m, 1H), 7.87 (dd, 1H), 8.65 (s, 1H).

Example 5 Preparation of4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-ol (Compound 0112) Step5a. 4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yl acetateHydrochloride (Compound 0111)

A mixture of 4-chloro-7-methoxyquinazolin-6-yl acetate (0105) (2.6 g,10.2 mmol) and 3-ethynylbenzenamine (0107) (2.4 g, 20.5 mmol) inisopropanol (100 ml) was stirred and heated to reflux for 3 hours. Themixture was cooled to room temperature. The precipitate was isolated anddried to give the title compound 0111 as a yellow solid (2.6 g, 68%):LCMS: m/z 334[M+1]⁺; ¹H NMR (DMSO) δ 2.39 (s, 3H), 3.17 (s, 1H), 3.98(s, 3H), 7.35 (m, 1H), 7.40 (s, 1H), 7.47 (m, 1H), 7.72 (m, 1H), 7.90(s, 1H), 8.57 (s, 1H), 8.87 (s, 1H), 10.99 (bs, 1H).

Step 5b. 4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-ol (Compound0112)

A mixture of compound 0111 (2.0 g, 5.4 mmol) and LiOH H₂O (0.75 g, 17.9mmol) in methanol (100 ml) and H₂O (100 ml) was stirred at roomtemperature for 0.5 hour. The mixture was neutralized by addition ofdilution acetic acid. The precipitate was isolated and dried to give thetitle compound 0112 as a grey solid (1.52 g, 96%): LCMS: m/z 292[M+1]⁺;¹H NMR (DMSO) δ 3.17 (s, 1H), 3.98 (s, 3H), 7.18 (d, 1H), 7.21 (s, 1H),7.37 (t, 1H), 7.80 (s, 1H), 7.90 (d, 1H), 8.04 (m, 1H), 8.47 (s, 1H),9.41 (s, 1H), 9.68 (bs, 1H).

Example 6 Preparation of Ethyl4-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)butanoate(Compound 0113-9) Step 6a. Ethyl4-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)butanoate(Compound 0113-9)

The title compound 0113-9 was prepared as a yellow solid (438 mg, 59%)from compound 0112 (500 mg, 1.72 mmol) and ethyl 4-bromobutyrate (349mg, 1.8 mmol) using a procedure similar to that described for compound0110-1 (Example 1): LCMS: m/z 406[M+1]⁺; ¹H NMR (CDCl₃) δ 1.37 (t, 3H),2.34 (m, 2H), 2.56 (t, 2H), 3.07 (s, 1H), 4.03 (s, 3H), 4.32 (m, 4H),7.21 (m, 1H), 7.25 (s, 1H), 7.36 (t, 1H), 7.94 (s, 1H), 7.97 (m, 1H),8.20 (s, 1H), 8.28 (m, 1H), 8.70 (s, 1H).

Example 7 Preparation of Ethyl6-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate(Compound 0113-11) Step 7a. Ethyl6-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate(Compound 0113-11)

The title compound 0113-11 was prepared as yellow solid (543 mg, 73%)from compound 0112 from step 5b (500 mg, 1.72 mmol) and ethyl6-bromohexanoate (401 mg, 1.8 mmol) using a procedure similar to thatdescribed for compound 0110-1 (Example 1): LCMS: m/z 434[M+1]⁺; ¹H NMR(CDCl₃) δ 1.24 (t, 3H), 1.53 (m, 2H), 1.72 (m, 2H), 1.90 (m, 2H), 2.37(t, 3H), 3.08 (s, 1H), 3.97 (s, 3H), 4.10 (m, 4H), 7.19 (s, 1H), 7.25(m, 2H), 7.34 (t, 1H), 7.67 (s, 1H), 7.78 (m, 1H), 7.84 (m, 1H), 8.67(s, 1H).

Example 8 Preparation of Ethyl6-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate(Compound 0113-12) Step 8a. Ethyl6-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate(Compound 0113-12)

The title compound 0113-12 was prepared as a yellow solid (305 mg, 84%)from compound 0112 from step 5b (247 mg, 0.85 mmol) and ethyl7-bromohepanoate (211 mg, 0.89 mmol) using a procedure similar to thatdescribed for compound 0110-1 (Example 1): LCMS: 448 [M+1]⁺; ¹H NMR(CDCl₃): δ1.15 (t, J=7.5 Hz, 3H), 1.33-1.60 (m, 6H), 1.81 (m, 2H), 2.28(t, J=7.5 Hz, 2H), 3.92 (s, 3H), 4.03 (q, J=7.2 Hz, 2H), 4.12 (t, J=6.6Hz, 2H), 4.18 (s, 1H), 7.19 (m, 2H), 7.39 (t, J=7.8 Hz, 1H), 7.80 (s,1H), 7.89 (d, J=8.1 Hz, 1H), 7.97 (s, 1H), 8.48 (s, 1H), 9.44 (s, 1H).

Example 8 Preparation of Ethyl6-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy) heptanoate(Compound 0408-12) Step 8a′. Ethyl 3-hydroxy-4-methoxybenzoate (Compound0402-12)

To a solution of ethyl 3,4-dihydroxybenzoate 0401 (12.52 g, 68.7 mmol)in DMF (50 mL) was added potassium carbonate (9.48 g, 68.7 mmol). Afterthe mixture was stirred for 15 minutes, a solution of iodomethane (9.755g, 68.7 mmol) in DMF (10 mL) was added dropwise. The reaction mixturewas stirred at 20° C. for 24 hours. After reaction the mixture wasfiltered, and the filtrate was concentrated. The residue was dissolvedin dichloromethane and washed with brine. The organic phase was driedover sodium sulfate, filtered and concentrated in vacuo to give crudeproduct. The crude product was purified by column chromatography to givethe title compound 0402-12 as a white solid (7.1 g, 53%): LCMS: 197[M+1]⁺, ¹H NMR (DMSO-d₆): δ 1.29 (t, J=6.6 Hz, 3H), 3.83 (s, 3H), 4.25(q, J=6.6 Hz, 2H), 7.00 (d, J=8.4 Hz, 1H), 7.38 (d, J=1.8 Hz, 1H), 7.43(dd, J=8.4 Hz, 2.1 Hz, 1H), 9.36 (s, 1H).

Step 8b′. Ethyl 3-(7-ethoxy-7-oxoheptyloxy)-4-methoxybenzoate (Compound0403-12)

A mixture of compound 0402-12 (6.34 g, 32.3 mmol), ethyl7-bromoheptanoate (7.66 g, 32.3 mmol) and potassium carbonate (13.38 g,96.9 mmol) in DMF (80 mL) was stirred at 60° C. for 3 hours. Afterreaction the mixture was filtrated. The filtrate was concentrated invacuo and the residue was dissolved in dichloromethane and washed withbrine twice. The organic phase was dried over sodium sulfate, filteredand concentrated to give the title product 0403-12 as a white solid(9.87 g, 86.7%): LCMS: 353 [M+1]⁺, ¹H NMR (DMSO-d₆): δ 1.17 (t, J=6.9Hz, 3H), 1.31 (t, J=7.2 Hz, 3H) 1.39 (m, 4H), 1.54 (m, 2H), 1.72 (m,2H), 2.29 (t, J=7.2 Hz, 2H), 3.83 (s, 3H), 3.98 (t, J=7.2 Hz, 2H), 4.06(q, J=6.9 Hz, 2H), 4.29 (q, J=7.2 Hz, 2H), 7.06 (d, J=8.4 Hz, 1H), 7.42(d, J=1.8 Hz, 1H), 7.57 (dd, J=8.4 Hz, 1.8 Hz, 1H).

Step 8c′. Ethyl 5-(7-ethoxy-7-oxoheptyloxy)-4-methoxy-2-nitrobenzoate(Compound 0404-12)

Compound 0403-12 (9.87 g, 28.0 mmol) was dissolved in acetic acid (20mL) and stirred at 20° C. Fuming nitric acid (17.66 g, 280.0 mmol) wasadded slowly dropwise. The mixture was stirred at 20° C. for 1 hour.After reaction the mixture was poured into ice-water and extracted withdichloromethane twice. The combined organic phase was washed with brine,aqueous NaHCO₃ solution and brine. The combined organic phase was driedover sodium sulfate, filtered and concentrated to give the title product0404-12 as a yellow solid (10.75 g, 96.4%): LCMS: 398 [M+1]⁺, ¹H NMR(DMSO-d₆): δ 1.17 (t, J=7.2 Hz, 3H), 1.27 (t, J=7.2 Hz, 3H), 1.38 (m,4H), 1.53 (m, 2H), 1.74 (m, 2H), 2.29 (t, J=7.2 Hz, 2H), 3.91 (s, 3H),4.03 (q, J=7.2 Hz, 2H), 4.08 (t, J=6.3 Hz, 2H), 4.30 (q, J=7.2 Hz, 2H),7.29 (s, 1H), 7.63 (s, 1H).

Step 8d′. Ethyl 2-amino-5-(7-ethoxy-7-oxoheptyloxy)-4-methoxybenzoate(Compound 0405-12)

A mixture of 0404-12 (10.75 g 27.0 mmol), ethanol (120 mL), water (40mL) and hydrogen chloride (4 mL) was stirred to form a clear solution.The iron powder (15.16 g, 27.0 mmol) was added batchwise. The mixturewas stirred at reflux for 30 min, and was then cooled to roomtemperature, adjusted pH to 8 with 10% sodium hydroxide solution, andfiltered. The filtrate was concentrated to remove ethanol and extractedwith dichloromethane twice. The combined organic phase was washed withbrine and dried over sodium sulfate, filtered and concentrated to givethe title product 0405-12 as a yellow solid (8.71 g, 87.8%): LCMS: 368[M+1]⁺, ¹H NMR (DMSO-d₆): δ 1.17 (t, J=7.2 Hz, 3H), 1.28 (t, J=7.2 Hz,3H), 1.37 (m, 4H), 1.53 (m, 2H), 1.66 (m, 2H), 2.29 (t, J=7.2 Hz, 2H),3.74 (s, 3H), 3.78 (t, J=6.9 Hz, 2H), 4.06 (q, J=7.2 Hz, 2H), 4.22 (q,J=7.2 Hz, 2H), 6.35 (s, 1H), 6.44 (s, 2H), 7.15 (s, 1H).

Step 8e′. Ethyl 7-(7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yloxy)heptanoate (Compound 0406-12)

A mixture of compound 0405-12 (8.71 g, 23.7 mmol), ammonium formate(1.48 g, 23.7 mmol) and formamide (40 mL) was stirred at 180° C. for 3hours. After reaction the mixture was cooled to room temperature. Theformamide was removed under reduce pressure, and the residue wasdissolved in dichloromethane and washed with brine. The organic phasewas dried over sodium sulfate, filtered and concentrated to give thetitle product 0406-12 as a pale white solid (8.18 g, 99%): LCMS: 349[M+1]⁺, ¹H NMR (DMSO-d₆): δ1.17 (t, J=6.9 Hz, 3H), 1.38 (m, 4H), 1.55(m, 2H), 1.75 (m, 2H), 2.29 (t, J=7.2 Hz, 2H), 3.90 (s, 3H), 4.05 (m,4H), 7.13 (s, 1H), 7.42 (s, 1H), 7.97 (d, J=3.6 Hz, 1H), 12.07 (s, 1H).

Step 8f′. Ethyl 7-(4-chloro-7-methoxyquinazolin-6-yloxy)heptanoate(Compound 0407-12)

A mixture of product 0406-12 (8.18 g, 23.5 mmol) and phosphoryltrichloride (50 mL) was stirred at reflux for 4 hours. After reactionthe excessive phosphoryl trichloride was removed under reduced pressure.The residue was dissolved in dichloromethane and washed with water,aqueous NaHCO₃ solution and brine. The organic phase was dried oversodium sulfate, filtered and concentrated to give the title product0407-12 as a yellow solid (5.93 g, 69.7%): LCMS: 367 [M+1]⁺, ¹H NMR(DMSO-d₆): δ 1.17 (t, J=6.9 Hz, 3H), 1.38 (m, 4H), 1.54 (m, 2H), 1.81(m, 2H), 2.30 (t, J=7.2 Hz, 2H), 4.02 (s, 3H), 4.06 (q, J=6.9 Hz, 2H),4.18 (t, J=6.3 Hz, 2H), 7.37 (s, 1H), 7.45 (s, 1H), 8.87 (s, 1H).

Step 8g′. Ethyl 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate (Compound 0408-12)

A mixture of product 0407-12 (5.93 g, 16.4 mmol) and3-ethynylbenzenamine (1.92 g, 16.4 mmol) in isopropanol (80 mL) wasstirred at reflux 4 hours. After reaction the mixture was cooled to roomtemperature and resulting precipitate was isolated, washed withisopropanol and ether, and dried to give the title compound 0408-12 as ayellow solid (4.93 g, 67.1%): LCMS: 448 [M+1]⁺, ¹H NMR (DMSO-d₆): δ 1.16(t, J=7.2 Hz, 3H), 1.36-1.59 (m, 6H), 1.80 (m, 2H), 2.29 (t, J=7.2 Hz,2H), 3.93 (s, 3H), 4.04 (q, J=6.9 Hz, 2H), 4.13 (t, J=6.6 Hz, 2H), 4.19(s, 1H), 7.20 (m, 2H), 7.39 (t, J=7.8 Hz, 1H), 7.81 (s, 1H), 7.89 (d,J=8.4 Hz, 1H), 7.97 (s, 1H), 8.48 (s, 1H), 9.45 (s, 1H).

Example 9 Preparation of Ethyl2-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)acetate (Compound0210-13) Step 9a. 6-Hydroxyquinazolin-4(3H)-one (compound 0202)

To a solution of 2-amino-5-hydroxybenzoic acid 0201 (30.6 g, 0.2 mol) informamide was stirred and heated to 190° C. for 0.5 h. The mixture wasallowed to cool to room temperature. The precipitate was isolated,washed with ether and dried to obtain title compound 0202 (32 g, brownsolid, yield: 99%): LC-MS m/z 163 [M+1];

¹H NMR (DMSO) δ 7.25 (dd, 1H), 7.40 (d, 1H), 7.46 (d, 1H), 7.88 (s, 1H).

Step 9b. 3,4-Dihydro-4-oxoquinazolin-6-yl acetate (Compound 0203)

A mixture of compound 0202 (30.0 g, 0.185 mol) and pyridine (35 ml) inacetic anhydride (275 ml) was stirred and heated at 100° C. for 2 hours.The reaction was poured into a mixture of ice and water (500 ml). Theprecipitate was isolated, washed with water and dried to obtain thetitle compound 0203 (24 g, pale white solid, yield: 61%): LC-MS m/z 205[M+1]; 1H-NMR (DMSO) δ 2.32 (s, 3H), 7.50 (dd, 1H), 7.80 (d, 1H), 7.98(s, 1H), 8.02 (s, 1H).

Step 9c. 4-Chloroquinazolin-6-yl acetate (Compound 0204)

A mixture of compound 0203 (20.0 g, 0.1 mol) in POCl₃ (150 ml) wasstirred and heated to reflux for 2 hours. The reaction was evaporatedand the residue was partitioned between ethyl acetate and a saturatedaqueous NaHCO₃ solution. The organic phase was washed with water, driedover Na₂SO₄ and evaporated. The mixture was purified by columnchromatography (silica gel, elution: 1:2=ethyl acetate/petroleum) toobtained the title compound 0204 (7.5 g, white solid, yield: 35%): LC-MSm/z 223 [M+1]; ¹H-NMR (CDCl₃) δ2.40 (s, 3H), 7.74 (dd, 1H), 8.00 (d,1H), 8.09 (d, 1H), 9.05 (s, 1H).

Step 9d. 4-(3-Chloro-4-fluorophenylamino)quinazolin-6-yl acetate(Compound 0207)

A mixture of 0204 (1.0 g, 4.5 mmol) and 3-chloro-4-fluorobenzenamine0205 (0.7 g, 5.0 mmol) in isopropanol (45 ml) was stirred and heated at90° C. for 1 hours. The reaction was cooled to room temperature and theprecipitate was isolated. The solid was washed in turn with isopropanoland methanol, dried to provide the title compound 0207 (1.3 g, paleyellow solid, yield: 87%): LC-MS m/z 332 [M+1]; 1H-NMR (DMSO) δ 2.37 (s,3H), 7.54 (t, 1H), 7.75 (m, 1H), 7.94 (dd, 1H), 7.99 (s, 1H), 8.02 (m,1H), 8.64 (s, 1H), 8.95 (s, 1H).

Step 9e. 4-(3-Chloro-4-fluorophenylamino)quinazolin-6-ol (Compound 0209)

A mixture of 0207 (0.8 g, 2.6 mmol) and lithium hydroxide monohydrate(0.13 g, 3.2 mmol) in methanol (10 ml)/water (15 ml) was stirred at roomtemperature for 1 hour. The pH was adjusted to 4 with acetic acid andfiltered. The collected yellow solid was washed by water and dried toobtained title compound 0209 (0.6 g, yellow solid, yield: 88%): LC-MSm/z 290 [M+1]; ¹H-NMR (DMSO) δ 7.42 (s, 1H), 7.45 (m, 1H), 7.70 (d, 1H),7.76 (s, 1H), 7.86 (m, 1H), 8.24 (q, 1H), 8.48 (s, 1H), 9.61 (s, 1H).

Step 9f. Ethyl2-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)acetate (Compound0210-13)

A mixture of 0209 (0.2 g, 0.77 mmol), ethyl 3-bromopropanoate (0.14 g,0.85 mmol) and K₂CO₃ (0.8 g, 5.8 mmol) in DMF (15 ml) was stirred andheated to 80° C. for 2 hours. The reaction was filtered and the filtratewas evaporated. The resulting solid was washed with ether to obtain thetitle compound 0210-13 (0.2 g, yellow solid, yield: 75%): mp 161-163°C.; LC-MS m/z 376 [M+1]; 1H-NMR (DMSO) δ 1.20 (t, 3H), 4.20 (q, 2H),4.96 (s, 2H), 7.45 (t, 1H), 7.55 (dd, 1H), 7.78 (m, 2H), 7.94 (d, 1H),8.16 (dd, 1H), 8.54 (s, 1H), 9.69 (s. 1H).

Example 10 Preparation of Ethyl4-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)-N-Hydroxybutanoate

The title compound was prepared (20 mg) from compound 0209 from step 9eand ethyl 4-bromobutanoate using a procedure similar to that describedfor Example 9: mp 128-132° C.; LC-MS m/z 391 [M+1]; ¹H-NMR (DMSO+D₂O) δ2.05 (m. 2H), 2.24 (t, 2H), 4.21 (t, 2H) 7.46 (t, 1H), 7.54 (dd, 1H),7.65 (m, 1H), 7.76 (d, 1H), 7.82 (m, 1H), 7.99 (m, 1H), 8.43 (s, 1H).

Example 11 Preparation of Ethyl6-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)hexanoate(compound 0210-17) Step 11a. Ethyl6-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)hexanoate(compound 0210-17)

The title compound 0210-17 (0.2 g) was prepared from compound 02094-(3-chloro-4-fluorophenylamino) quinazolin-6-ol and ethyl6-bromohexanoate using a procedure similar to that described forcompound 0210-13 (Example 9): LC-MS m/z 433 [M+1], ¹H-NMR (DMSO) δ 1.13(t, 3H), 1.45 (m, 2H), 1.60 (m, 2H), 1.76 (m, 2H), 2.30 (t, 2H), 4.05(q, 2H), 4.11 (t, 2H), 7.41 (d, 1H), 7.45 (dd, 1H), 7.68 (d, 1H), 7.80(m, 1H), 7.86 (m, 1H), 8.13 (dd, 1H), 8.48 (s, 1H).

Example 12 Preparation of Ethyl7-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)heptanoate(Compound 0210-18) Step 12a. Ethyl7-(4-(3-chloro-4-fluorophenylamino)quinazolin-6-yloxy)heptanoate(Compound 0210-18)

The title compound 0210-18 (0.2 g) was prepared from compound 2-64-(3-chloro-4-fluorophenylamino)quinazolin-6-ol (0209) of step 9e andethyl 7-bromoheptanoate using a procedure similar to that described forcompound 0210-13 (Example 9): LC-MS m/z 420 [M+1], ¹H-NMR (DMSO) δ 1.13(t, 3H), 1.36 (m, 2H), 1.46 (m, 2H), 1.54 (m, 2H), 1.78 (m, 2H), 2.27(t, 2H), 4.05 (q, 2H), 4.11 (t, 2H), 7.41 (d, 1H), 7.47 (dd, 1H), 7.70(d, 1H), 7.81 (m, 1H), 7.84 (m, 1H), 8.13 (dd, 1H), 8.50 (s, 1H).

Biological Assays:

As stated hereinbefore the derivatives defined in the present inventionpossess anti-proliferation activity. These properties may be assessed,for example, using one or more of the procedures set out below:

(a) An In Vitro Assay which Determines the Ability of a Test Compound toInhibit EGFR Kinase.

The ability of compounds to inhibit receptor kinase (EGFR) activity canbe assayed using HTSCAN™ EGF Receptor Kinase Assay Kits (Cell SignalingTechnologies, Danvers, Mass.). EGFR tyrosine kinase is obtained asGST-kinase fusion protein which is produced using a baculovirusexpression system with a construct expressing human EGFR(His672-Ala1210) (GenBank Accession No. NM_(—)005228) with anamino-terminal GST tag. The protein is purified by one-step affinitychromatography using glutathione-agarose. An anti-phosphotyrosinemonoclonal antibody, P-Tyr-100, is used to detect phosphorylation ofbiotinylated substrate peptides (EGFR, Biotin-PTP1B (Tyr66)). Enzymaticactivity is tested in 60 mM HEPES, 5 mM MgCl₂ 5 mM MnCl₂ 200 μM ATP,1.25 mM DTT, 3 μM Na₃VO₄, 1.5 mM peptide, and 50 ng EGF Receptor Kinase.Bound antibody is detected using the DELFIA system (PerkinElmer,Wellesley, Mass.) consisting of DELFIA® Europium-labeled Anti-mouse IgG(PerkinElmer, #AD0124), DELFIA® Enhancement Solution (PerkinElmer,#1244-105), and a DELFIA® Streptavidin coated, 96-well Plate(PerkinElmer, AAAND-0005). Fluorescence is measured on a WALLAC Victor 2plate reader and reported as relative fluorescence units (RFU). Data canbe plotted using GraphPad Prism (v4.0a) and IC50's calculated using asigmoidal dose response curve fitting algorithm.

Test compounds are dissolved in dimethylsulphoxide (DMSO) to give a 20mM working stock concentration. Each assay is setup as follows: Added100 μl of 10 mM ATP to 1.25 ml 6 mM substrate peptide. Dilute themixture with dH₂0 to 2.5 ml to make 2×ATP/substrate cocktail ([ATP]=400mM, [substrate]=3 mM). Immediately transfer enzyme from −80° C. to ice.Allow enzyme to thaw on ice. Microcentrifuge briefly at 4° C. to bringliquid to the bottom of the vial. Return immediately to ice. Add 10 μlof DTT (1.25 mM) to 2.5 ml of 4×HTScan™ Tyrosine Kinase Buffer (240 mMHEPES pH 7.5, 20 mM MgCl₂, 20 mM MnCl, 12 mM NaVO₃) to make DTT/Kinasebuffer. Transfer 1.25 ml of DTT/Kinase buffer to enzyme tube to make 4×reaction cocktail ([enzyme]=4 ng/μL in 4× reaction cocktail). Incubate12.5 μl of the 4× reaction cocktail with 12.5 μl/well of predilutedcompound of interest (usually around 10 μM) for 5 minutes at roomtemperature. Add 25 μl of 2×ATP/substrate cocktail to 25 μl/wellpreincubated reaction cocktail/compound. Incubate reaction plate at roomtemperature for 30 minutes. Add 50 μl/well Stop Buffer (50 mM EDTA, pH8) to stop the reaction. Transfer 25 μl of each reaction and 75 μldH₂O/well to a 96-well streptavidin-coated plate and incubated at roomtemperature for 60 minutes. Wash three times with 200 μl/well PBS/T(PBS, 0.05% Tween-20). Dilute primary antibody, Phospho-Tyrosine mAb(P-Tyr-100), 1:1000 in PBS/T with 1% bovine serum albumin (BSA). Add 100μl/well primary antibody. Incubate at room temperature for 60 minutes.Wash three times with 200 μl/well PBS/T. Dilute Europium labeledanti-mouse IgG 1:500 in PBS/T with 1% BSA. Add 100 μl/well dilutedantibody. Incubate at room temperature for 30 minutes. Wash five timeswith 200 μl/well PBS/T. Add 100 μl/well DELFIA® Enhancement Solution.Incubate at room temperature for 5 minutes. Detect 615 nm fluorescenceemission with appropriate Time-Resolved Plate Reader.

(b) An In Vitro Assay which Determines the Ability of a Test Compound toInhibit the EGF-Stimulated EGFR Phosphorylation.

Allow A431 cell growth in a T75 flask using standard tissue cultureprocedures until cells reach near confluency (˜1.5×10⁷) cells; D-MEM,10% FBS). Under sterile conditions dispensed 100 μl of the cellsuspension per well in 96-well microplates (x cells plated per well).Incubate cells and monitor cell density until confluency is achievedwith well-to-well consistency; approximately three days. Remove completemedia from plate wells by aspiration or manual displacement. Replacemedia with 50 μl of pre-warmed serum free media per well and incubated 4to 16 hours. Make two fold serial dilutions of inhibitor usingpre-warmed D-MEM so that the final concentration of inhibitor range from10 μM to 90 pM. Remove media from A431 cell plate. Add 100 μl of serialdiluted inhibitor into cells and incubate 1 to 2 hours. Remove inhibitorfrom plate wells by aspiration or manual displacement. Add either serumfree media for resting cells (mock) or serum free media with 100 ng/mlEGF. Use 100 μl of resting/activation media per well. Allow incubationat 37° C. for 7.5 minutes. Remove activation or stimulation mediamanually or by aspiration. Immediately fix cells with 4% formaldehyde in1×PBS. Allow incubation on bench top for 20 minutes at RT with noshaking Wash five times with 1×PBS containing 0.1% Triton X-100 for 5minutes per Wash. Remove Fixing Solution. Using a multi-channelpipettor, add 200 μl of Triton Washing Solution (1×PBS+0.1% TritonX-100). Allow wash to shake on a rotator for 5 minutes at roomtemperature. Repeat washing steps 4 more times after removing washmanually. Using a multi-channel pipettor, block cells/wells by adding100 μl of LI-COR Odyssey Blocking Buffer to each well. Allow blockingfor 90 minutes at RT with moderate shaking on a rotator. Add the twoprimary antibodies into a tube containing Odyssey Blocking Buffer. Mixthe primary antibody solution well before addition to wells(Phospho-EGFR Tyr1045, Rabbit; 1:100 dilution; Cell SignalingTechnology, 2237; Total EGFR, Mouse; 1:500 dilution; BiosourceInternational, AHR5062). Remove blocking buffer from the blocking stepand added 40 μl of the desired primary antibody or antibodies in OdysseyBlocking Buffer to cover the bottom of each well. Add 100 μl of OdysseyBlocking Buffer only to control wells. Incubate with primary antibodyovernight with gentle shaking at RT. Wash the plate five times with1×PBS+0.1% Tween-20 for 5 minutes at RT with gentle shaking, using agenerous amount of buffer. Using a multi-channel pipettor add 200 μl ofTween Washing Solution. Allow wash to shake on a rotator for 5 minutesat RT. Repeat washing steps 4 more times. Dilute the fluorescentlylabeled secondary antibody in Odyssey Blocking Buffer (Goat anti-mouseIRDye™ 680 (1:200 dilution; LI-COR Cat.#926-32220) Goat anti-rabbitIRDye™ 800CW (1:800 dilution; LI-COR Cat.#926-32211). Mix the antibodysolutions well and added 40 μl of the secondary antibody solution toeach well. Incubate for 60 minutes with gentle shaking at RT. Protectplate from light during incubation. Wash the plate five times with1×PBS+0.1% Tween-20 for 5 minutes at RT with gentle shaking, using agenerous amount of buffer. Using a multi-channel pipettor add 200 μl ofTween Washing Solution. Allow wash to shake on a rotator for 5 minutesat RT. Repeat washing steps 4 more times. After final wash, remove washsolution completely from wells. Turned the plate upside down and tap orblot gently on paper towels to remove traces of wash buffer. Scan theplate with detection in both the 700 and 800 channels using the OdysseyInfrared Imaging System (700 nm detection for IRDye™ 680 antibody and800 nm detection for IRDye™ 800CW antibody). Determine the ratio oftotal to phosphorylated protein (700/800) using Odyssey software andplot the results in Graphpad Prism (V4.0a). Data can be plotted usingGraphPad Prism (v4.0a) and IC50's calculated using a sigmoidal doseresponse curve fitting algorithm.

(c) An In Vitro Assay which Determines the Ability of a Test Compound toInhibit HDAC Enzymatic Activity.

HDAC inhibitors are screened using an HDAC fluorimetric assay kit(AK-500, Biomol, Plymouth Meeting, Pa.). Test compounds can be dissolvedin dimethylsulphoxide (DMSO) to give a 20 mM working stockconcentration. Fluorescence is measured on a WALLAC Victor 2 platereader and reported as relative fluorescence units (RFU). Data areplotted using GraphPad Prism (v4.0a) and IC50's calculated using asigmoidal dose response curve fitting algorithm.

Each assay is setup as follows: Defrost all kit components and keep onice until use. Dilute HeLa nuclear extract 1:29 in Assay Buffer (50 mMTris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2). Prepare dilutionsof Trichostatin A (TSA, positive control) and test compounds in assaybuffer (5× of final concentration). Dilute Fluor de Lys™ Substrate inassay buffer to 100 uM (50 fold=2× final). Dilute Fluor de Lys™developer concentrate 20-fold (e.g. 50 μl plus 950 μl Assay Buffer) incold assay buffer. Second, dilute the 0.2 mM Trichostatin A 100-fold inthe 1× Developer (e.g. 10 μl in 1 ml; final Trichostatin A concentrationin the 1× Developer=2 μM; final concentration after addition toHDAC/Substrate reaction=1 μM). Add Assay buffer, diluted trichostatin Aor test inhibitor to appropriate wells of the microtiter plate. Adddiluted HeLa extract or other HDAC sample to all wells except fornegative controls. Allow diluted Fluor de Lys™ Substrate and the samplesin the microtiter plate to equilibrate to assay temperature (e.g. 25 or37° C. Initiate HDAC reactions by adding diluted substrate (25 μl) toeach well and mixing thoroughly.

Allow HDAC reactions to proceed for 1 hour and then stopped them byaddition of Fluor de Lys™ Developer (50 μl). Incubate plate at roomtemperature (25° C.) for 10-15 min. Read samples in a microtiter-platereading fluorimeter capable of excitation at a wavelength in the range350-380 nm and detection of emitted light in the range 440-460 nm.

The following compounds in TABLE B were screened in assays substantiallyas described above. The results are presented below. In these assays,the following grading was used: I≧10 μM, 10 μM>II>1 μM, 1 μM>III>0.1 μM,and IV≦0.1 μM for IC₅₀.

TABLE B IC50 (nM) Structure HDAC EGFR HER2/ErbB2

IV

I IV III

I IV

I

Cell Proliferation Assay:

Cancer cell lines are plated at 5,000 to 10,000 per well in 96-wellflatted bottomed plates with various concentration of compounds. Thecells are incubated with compounds for 72 hours in the presence of 0.5%of fetal bovine serum. Growth inhibition is accessed by adenosinetriphosphate (ATP) content assay using Perkin Elmer ATPlite kit. ATPliteis an ATP monitoring system based on firefly luciferase. Briefly, 25 μlof mammalian cell lysis solution is added to 50 μl of phenol red-freeculture medium per well to lyse the cells and stabilize the ATP. 25 μlof substrate solution is then added to the well and subsequently theluminescence is measured.

Tumor cell lines that can be assayed include those listed in TABLES Cand D.

TABLE C Cell Line Breast_MCF7 Breast_MDAMB468 Breast_SkBr3 Colon_HCT116Epidermoid_A431 Lung_H1703 Lung_H1975 Lung_H2122 Lung_H292 Lung_H358Lung_H460 Lung_HCC827 Pancreas_BxPC3 Pancreas_Capan1 Pancreas_CFPACPancreas_HPAC Pancreas_MiaPaCa2 Pancreas_PANC1 Prostate_22RV1Prostate_PC3

TABLE D Model Cancer type A431 Epidermoid H358 NSCLC H292 NSCLC BxPC3Pancreatic PC3 Prostate HCT116 Colon HCC827(apoptosis/anti-proliferation) NSCLC BxPC3(apoptosis/anti-proliferation) Pancreatic

A representative protocol for the in vivo experiment is as followed:

1-10×10⁶ human cancer cells are implanted subcutaneously to the athymic(nu/nu) mice. When the tumors reach about 100 mm³ in volume, the miceare treated with the compound by tail vein infusion. Routinely 5 groups(8-12 mice per group) are needed for a typical efficacy study, includingone negative control, one positive control, and three testing groups for3 dose levels of the same compound. Usually a 7-7-5 (on-off-on) regimenis used for one typical study. The tumor size is measured with anelectronic caliper and body weight measured with a scale twice weekly.The tumors are removed from euthanized mice at the end of the study. Onehalf of each tumor is frozen in dry ice and stored at −80° C. for PK orWestern blot analysis. The other half is fixed with formalin. The fixedtissues are processed, embedded in paraffin and sectioned forimmunohistochemistry staining.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of inhibiting epidermal growth factortyrosine kinase activity in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acompound represented by formula (I):

or a pharmaceutically acceptable salt or ester thereof, wherein X is O,S, CH₂, or CONH; B is an unsubstituted C₃ to C₉ alkylene; R₁ is selectedfrom hydrogen, hydroxy, and C₁ to C₄ alkoxy; each R₂ is independentlyselected from halogen, hydroxy, C₁ to C₄ alkyl, C₂ to C₄ alkenyl, and C₂to C₄ alkynyl; and n is 1, 2 or
 3. 2. The method of claim 1, wherein thecompound of formula (I) is represented by formula (II):

or a pharmaceutically acceptable salt or ester thereof.
 3. The method ofclaim 2, wherein B is a straight chain C₅ to C₇ alkylene.
 4. The methodof claim 3, wherein B is a straight chain C₆ alkylene.
 5. The method ofclaim 2, wherein the compound of formula (II) is represented by formula(III):


6. The method of claim 5, wherein B is a straight chain C₅ to C₇alkylene.
 7. The method of claim 6, wherein R₁ is hydroxy or methoxy andB is a straight chain C₆ alkylene.
 8. The method of claim 2, wherein thecompound of formula (II) is represented by formula (IV):


9. The method of claim 8, wherein B is a straight chain C₅ to C₇alkylene.
 10. The method of claim 9, wherein R₁ is hydroxy or methoxyand B is a straight chain C₆ alkylene.
 11. The method of claim 1,wherein the compound of formula (I) is represented by formula (V):


12. The method of claim 11, wherein B is a straight chain C₅ to C₇alkylene.
 13. The method of claim 12, wherein R₁ is hydroxy or methoxyand B is a straight chain C₆ alkylene.
 14. The method of claim 1,wherein the subject has an epidermal growth factor receptor tyrosinekinase related cancer.
 15. The method of claim 14, wherein the epidermalgrowth factor receptor tyrosine kinase related cancer is selected fromthe group consisting of papilloma, glioblastoma, Kaposi's sarcoma,melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladdercancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer,pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia,lymphoma, Hodgkin's disease and Burkitt's disease.
 16. The method ofclaim 1, wherein B is an unsubstituted straight chain C₆ alkylene. 17.The method of claim 1, wherein R₁ is selected from hydrogen, hydroxy andmethoxy.
 18. The method of claim 1, wherein each R₂ is independentlyselected from Br, Cl, F, hydroxy and ethynyl.
 19. The method of claim 1wherein n is 1 or
 2. 20. The method of claim 1, wherein the compound isof the formula:

or a pharmaceutically acceptable salt or ester thereof.